Abstract Today, 25 years after the first umbilical cord blood transplant (UCB), an estimated 800,000 UCB units have been banked and >30,000 UCB units have been used for transplantation of both adults and children. UCB has addressed some of the most pressing obstacles of allogeneic HSCT, namely, rapid access and HLA match requirement, but it too has its challenges, namely, limited cell dose and consequent delays in lympho- hematopoietic recovery. Since the first transplant for leukemia in 1990 by the PPG PI, we have performed >1400 UCBT, with more transplants in adults than any another institution. Through PPG sponsorship, we have exploited the benefits and tackled the limitations of UCB. Among the key discoveries are: 1) identification of the CD34 cell dose threshold, 2) development of the double UCBT platform as a powerful tool to evaluate graft engineering strategies in addition to its general use for patients without an adequate single unit, 3) optimization of conditioning regimens tailored to UCBT, and 4) development of novel cellular therapeutics, namely UCB- derived T regulatory cells (Treg) for prevention of acute graft-versus-host disease (GVHD), and, StemRegenin- 1 expanded UCB CD34+ cells to accelerate neutrophil and platelet recovery. While we have made substantial progress, slow immune recovery and high risk of infection persists. Therefore, the overarching aim of Project 1 is to accelerate lympho-hematopoietic recovery and Immune reconstitution. Building upon past discoveries, we will tackle prolonged immunoincompetence in 3 ways: 1) We will determine the impact of high UCB CD34+ cell doses on risk of infection and immune reconstitution (aim 1). During the current funding period, we showed StemRegenin-1 (SR-1) enabled massive expansion of UCB HSC/progenitors, resulting in a significant reduction in the period of cytopenia after UCBT. Having demonstrated its long-term engraftment potential when co-infused with an unmanipulated 'back-up' unit, SR -1 expanded UCB will now be evaluated as a 'stand-alone' graft. 2) We will determine the essential ratio of T effector to Treg that will reliably prevent GVHD and determine its effect on risk of infection and immune reconstitution (aim 2). During the current funding period, we developed a robust method for manufacturing billions of CD4+25+FoxP3+127- cells from a single UCB unit and demonstrated its ability to completely abrogate acute GVHD, a dramatic departure from the historical expectation of 48% in older adults. 3) We will determine the safety and efficacy of UCB-derived thymic progenitors (Tprog) that have been shown to repair damaged thymic epithelial cells and contribute to immune recovery in 'proof of concept' animal models (aim 3). During the current funding period, we successfully developed a culture expansion strategy in support of a 'first-in-human' phase I clinical trial. We believe that this comprehensive, multi-dimensional approach will have a marked impact the pace of immune recovery and risk of infection - among the greatest challenges facing transplant medicine today.